The broad objective of this project is the detailed molecular characterization of the Na+/l- symporter (NIS), a key intrinsic plasma membrane transport protein that mediates active translocation of I- into the thyroid. NIS plays a crucial role in thyroid hormogenesis and in the diagnosis and treatment of thyroid diseases, such as the use of radioiodide therapy in thyroid cancer. Starting with the isolation of the NIS cDNA earlier in this project, much progress has been made in the characterization of the protein at all levels, including the identification of functionally important residues through the characterization of NIS mutations that cause I- transport defect. NIS also mediates I- transport in other tissues, including lactating breast and breast cancer metastases, so NIS is of potential therapeutic significance in breast cancer. By means of gene transfer, NIS may also be valuable in the treatment of cancer in other tissues that do not express NIS endogenously. The studies proposed here emphasize the key process of trafficking of NIS to and from the plasma membrane, the elucidation of which is of major basic and medical relevance. The following specific aims are proposed: 1. To elucidate the molecular mechanism by which I- regulates its own NIS-mediated transport: a) does I- at high concentrations affect NIS degradation? b) what is the NIS degradation pathway? c) what is the pathway of I- -induced NIS internalization? d) does I- -induced NIS dimerization occur by intermolecular disulfide bond formation? e) is the thyroid Duox oxidase involved in the inhibition of NIS-mediated I- transport by I-? 2. To identify the determinants for polarized trafficking of NIS to the basolateral plasma membrane: a) what are the signals within the NIS Ct that target the protein to the basolateral membrane? b) can the determinants of NIS basolateral targeting reverse the targeting of the sodium/monocarboxylate transporter (SMCT) from apical to basolateral? c) is the PDZ-domain-containing protein hScrib involved in NIS targeting to and/or retention at the plasma membrane? 3. To establish new structure/function relations in NIS: we will a) ascertain the role played by amino acid residues present in transmembrane segment (TMS) IX in Na+ dependence;b) perform functional analyses of chimera NIS/SMCT proteins to assess the role of functionally important regions and/or amino acid residues in the Na+ and I- translocation pathways, as well as in Na+/l- coupling;c) undertake the solubilization, reconstitution, and purification of NIS expressed in E. coli.